Isolated plasma membranes from Saccharomyces cerevisiae were shown to catalyze unidirectional synthesis of chitin. The polysaccharide is laid down on the external surface of the membrane, whereas chitin synthetase appears to face the cytoplasm. It is concluded that in vitro as well as in vivo the synthetase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfer them to a growing chain of chitin that is simultaneously extruded to the outside. This is the first demonstration of vectorial synthesis of a polysaccharide by isolated membranes. A novel step in the purification of chitin synthetase, entrapment by the product of the reaction, i.e., by chitin, has been refined and now results in a 100-fold purification with 50% yield. The purified enzyme is still able to form chitin after electrophoresis on acrylamide gel. Its position on the gel can thus be established. Preliminary experiment on the synthesis of glucan and its regulation in Wangiella dermatitidis have been started.